Glucolipotoxic stress-activated NR4A1 shifts MSC mitochondrial transfer to MCOLN1–mTORC1-dependent exocytosis and compromises proangiogenic support

Glucolipotoxicity-induced dysfunction of mesenchymal stem cells (MSCs) limits their therapeutic efficacy in diabetic vascular complications, yet the underlying mechanisms remain poorly understood. Emerging evidence identifies mitochondrial transfer, the direct delivery of functional mitochondria from MSCs to recipient cells, as an important therapeutic mechanism. However, the regulatory mechanisms and functional significance of MSC-mediated mitochondrial transfer under such conditions remain unclear. Thus, elucidating how glucolipotoxicity impairs MSC-mediated mitochondrial transfer and angiogenesis may inform strategies to restore this supportive function. In vitro, we assessed MSC support for endothelial cells (ECs) under high-glucose and palmitate (HG/PAL) conditions by measuring EC migration, proliferation, and apoptosis. NR4A1 function was examined by siRNA/shRNA knockdown and agonist stimulation. We then analysed mitochondrial function (ROS, membrane potential) in HG/PAL-treated MSCs and examined the regulatory role of NR4A1. Mitochondrial transfer and exocytosis were assessed by flow cytometry, with exocytosis further confirmed by electron microscopy. Bioinformatic prediction, ChIP-qPCR, and Western blot were used to delineate the NR4A1–MCOLN1–mTORC1–TFEB pathway that regulates mitochondrial exocytosis. In vivo, Matrigel plug assays in nude mice were used to evaluate MSC-driven angiogenesis and mitochondrial transfer. NR4A1 was upregulated in MSCs under HG/PAL, and its knockdown rescued MSC proliferation, migration, and capacity to support EC function. Mechanistically, NR4A1 disrupted mitochondrial dynamics and impaired lysosomal autophagic flux via mTORC1 activation, triggering mitochondrial exocytosis that depleted the intracellular mitochondrial pool and reduced mitochondrial transfer to ECs. This effect was partially counterbalanced by an NR4A1–MCOLN1 negative feedback loop that limits mTORC1 hyperactivation. In vivo, NR4A1 knockdown restored PAL-treated MSC-driven angiogenesis and mitochondrial transfer. Our findings establish NR4A1 as a molecular switch that redirects MSCs from mitochondrial donors to pathological exporters, providing both a mechanistic basis and a therapeutic target to preserve MSC function under glucolipotoxic stress.